Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The early steps in the transposition of Mu DNA have been examined in vitro using purified Mu A, Mu B and E. coli HU proteins, a mini-Mu donor plasmid and target DNA. Two stable protein-DNA complexes, or transpososomes, have been characterized. Type 1 complex formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein. In the Type 1 complex the two ends of Mu are held together, creating two independent topological domains; a supercoiled Mu domain and a relaxed vector domain (due to nicking at the Mu ends). In the presence of Mu B protein, ATP, and target DNA, the Type 1 complex is converted into a Type 2 complex which is the protein associated product of the strand transfer reaction.;Type 1 complex formation with a mini-Mu plasmid with in vivo level of supercoiling ({dollar}\sigma{dollar} = {dollar}-{dollar}0.025) was dependent on an additional E. coli protein factor which we named supercoiling relief factor (SRF). This protein was shown to be integration host factor (IHF). Low levels of IHF also reduce the amount of HU and Mu A required for the reaction and at high concentrations of IHF, HU is not required. The SRF activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which stimulates the initial rate of the in vitro reaction 100-fold.;Point mutations at the terminal nucleotide of the Mu ends in a mini-Mu plasmid inhibit the introduction of nicks at the Mu ends in a reaction with Mu A, HU and IHF proteins. Addition of Mu B and ATP, however, dramatically stimulates the reaction of mutant mini-Mu plasmids carrying the mutation at one end. Two products are observed; a normal Type 1 complex which contains nicks at both Mu ends, and free relaxed plasmid which is nicked only at the wild type end. Stable protein-DNA complexes characteristic of the first step in the in vitro transposition reaction require the introduction of nicks at both Mu ends.



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